Bottom Line:
On the basis of our previous discovery of a set of nine naturally occurring amino acid substitutions that functionally interconverted orthologous sesquiterpene synthases from Nicotiana tabacum and Hyoscyamus muticus, we created a library of all possible residue combinations (2(9) = 512) in the N. tabacum enzyme.The product spectra of 418 active enzymes revealed a rugged landscape where several minimal combinations of the nine mutations encode convergent solutions to the interconversions of parental activities.These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.

Figure 5: Biosynthetic tree of the M9 libraryA similarity-based cluster diagram was constructed to quantitatively organize the M9 library according to terpene product spectra from the pair-wise alignment of product proportions for each of the 418 active mutants (described in Methods). Clades are colored according to the major reaction product (defined in Fig. 4a), with representative chromatograms identified and numbered branching off each major clade. Product peaks in the chromatograms are colored blue (5-EA, 2), purple (4-EE, 4) or red (PSD, 3).

Mentions:
To quantitatively examine the distribution of biosynthetic activities across the M9 library, we performed a sum of least squares pairwise comparison of chemical product proportions. The resulting ‘chemical’ distance matrix was condensed to produce an un-rooted neighbor-joining “biosynthetic tree” (Fig. 5). This tree shows several distinct clusters or clades separating TEAS and HPS-like activities at either end. Annotating each clade with chromatograms from representative mutants highlights the common product profiles that define its members. For example, a promiscuous clade near the tree center is marked by elevated production of germacrene A (5) in mutant “(8)”. Sequence analysis of the HPS-like clade reveals a clustering of mutants into distinct groups based on sequence, indicating that several convergent solutions exist along a subset of synthetic lineages (Supplementary Fig. 2 online). By comparison, members of the EES-like clade generally possess diverse sequences but exhibit a strict dependence on the T402S active site mutation for EES activity.

Figure 5: Biosynthetic tree of the M9 libraryA similarity-based cluster diagram was constructed to quantitatively organize the M9 library according to terpene product spectra from the pair-wise alignment of product proportions for each of the 418 active mutants (described in Methods). Clades are colored according to the major reaction product (defined in Fig. 4a), with representative chromatograms identified and numbered branching off each major clade. Product peaks in the chromatograms are colored blue (5-EA, 2), purple (4-EE, 4) or red (PSD, 3).

Mentions:
To quantitatively examine the distribution of biosynthetic activities across the M9 library, we performed a sum of least squares pairwise comparison of chemical product proportions. The resulting ‘chemical’ distance matrix was condensed to produce an un-rooted neighbor-joining “biosynthetic tree” (Fig. 5). This tree shows several distinct clusters or clades separating TEAS and HPS-like activities at either end. Annotating each clade with chromatograms from representative mutants highlights the common product profiles that define its members. For example, a promiscuous clade near the tree center is marked by elevated production of germacrene A (5) in mutant “(8)”. Sequence analysis of the HPS-like clade reveals a clustering of mutants into distinct groups based on sequence, indicating that several convergent solutions exist along a subset of synthetic lineages (Supplementary Fig. 2 online). By comparison, members of the EES-like clade generally possess diverse sequences but exhibit a strict dependence on the T402S active site mutation for EES activity.

Bottom Line:
On the basis of our previous discovery of a set of nine naturally occurring amino acid substitutions that functionally interconverted orthologous sesquiterpene synthases from Nicotiana tabacum and Hyoscyamus muticus, we created a library of all possible residue combinations (2(9) = 512) in the N. tabacum enzyme.The product spectra of 418 active enzymes revealed a rugged landscape where several minimal combinations of the nine mutations encode convergent solutions to the interconversions of parental activities.These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.